Probiotic strains, a process for the selection of them, compositions thereof, and their use

ABSTRACT

The present invention relates to a novel process for the selection of new probiotic strains which comprises the following steps: a) selecting for non-pathogenic strains which are capable of surviving in breast milk and/or amniotic fluid, and b) selecting for non-pathogenic strains which are able to be transferred to breast milk and/or amniotic fluid after oral intake in healthy individuals without colonizing other internal organs except mucousas. The invention also provides new Lactobacillus strains, which are: CECT5711 ( Lactobacillus coryniformis ), CECT5713 ( Lactobacillus salivarius  subsp. salivarius), CECT5714: ( Lactobacillus gasseri , formerly  L. acidophilus ), CETC5715: ( Lactobacillus gasseri ), and CECT5716: ( Lactobacillus fermentum ); and refers to their use for the prophylaxis or treatment against digestive, infective, neuro-degenerative and immune related diseases such as allergies or inflammatory diseases.

FIELD OF THE INVENTION

[0001] The present invention relates to a novel process for theselection of new probiotic strains, to new probiotic microorganisms ofthe genus Lactobacillus selected according to this method and tocompositions comprising these microorganisms; to their use for theprophylaxis or treatment against digestive, infective,neuro-degenerative and immune related diseases such as allergies orinflammatory diseases, and to a novel source to obtain saidmicroorganisms.

BACKGROUND OF THE INVENTION

[0002] For many years, lactic acid bacteria have been utilized asfermenting agents for the preservation of food taking benefit of a lowpH and the action of fermentation products generated during thefermentative activity thereof to inhibit the growth of spoilagebacteria. With this aim, non-well characterized lactic acid bacteria or“fermentum” have been used to prepare a variety of different foodstuffssuch as dry fermented meat products, cheese, and other fermented dairyproducts from milk.

[0003] Recently, lactic acid bacteria have attracted a great deal ofattention because some strains have been found to exhibit valuableproperties to man and animals upon ingestion. In particular, specificstrains of the genus Lactobacillus or Bifidobacterium have been found tobe able to colonize the intestinal mucosa and to assist in themaintenance of the well-being of man and animal, and has been named suchas probiotics.

[0004] Probiotics are considered to be viable microbial preparationswhich promote the individual's health by preserving a healthiermicroflora in the intestine. A microbial preparation may be commonlyaccepted as a probiotic in case the effectual thereof and their mode ofaction are known. Probiotics are deemed to attach to the intestine'smucosa, colonize the intestinal tract and likewise prevent attachment ofharmful microorganisms thereon. A crucial prerequisite for their actionresides in that they have to reach the gut's mucosa in a proper andviable form and do not get destroyed in the upper part of thegastrointestinal tract, especially by the influence of the low pHprevailing in the stomach.

[0005] During the extensive studies leading to new probiotic strains,previous patent applications have described the isolation of variety ofdifferent bacterial strains from baby feces (JP04320642, JP05227946).Moreover, the probiotic strains obtained up to now were mainly selectedfor their capability to adhere to the intestinal mucosa, usually by invitro experiments. Subsequent selection has not always been performed,and if it has, it used to be mainly based on individual properties ofthe strain. Finally, and sometimes after the commercialization of thestrain, the beneficial effects of the selected strain have been provenin vivo.

[0006] In this regard, several patent applications such as eg.EP0768375, WO97/00078, EP0577903 and WO00/53200 disclose specificstrains of Bifidibacterium and Lactobacillus and their beneficialeffects on diarrhea, immunomodulation, hypersensitivity reactions orinfection by pathogen microorganisms.

[0007] Moreover, the beneficial effects of human breast milk on thewell-being of infants compared to those fed with milk-based formula hasalso been extensively reported. In this regard, a reduction in the riskof infection, allergy, asthma and related affections, and an improvementof the intestinal maturation and gut functions has been described. Also,it has been reported that the composition of the gut flora is differentto human-milk fed infants from those fed with milk-based formula. Thebeneficial effects and the modulation of the gut flora of breast humanmilk have been attributed to its characteristic composition as comparedto infant formulas. Thus, the benefits of breast milk proteins such aslactoferrin or maternal immunoglobulins, and the rich composition inoligosaccharides that may act as prebiotic compounds, in the regulationof the flora and gut functions have been reported.

[0008] However, to our knowledge there is no publication or work thatdescribes the presence of microbial strains in normal human breast milk.Neither it has been reported that such microbial strains could bebeneficial for the breast-fed baby, and therefore acting as probioticsmodulating the gut flora of the breast feeding infant. Our work suggeststhat the well-being effects of the human breast-fed could be alsomediated by microbial strains present therein.

[0009] It has been suggested in several works that the initialcolonization of the neonate is due to cross-contamination with vaginalmicroflora during labor. However, there are several studies that showsimilar initial microbial colonization of the neonate independently ofthe neonate delivery route (cesarean versus natural labor). Moreover,the fact that it is not possible to obtain germ-free animals fromconventional pregnant mice nevertheless they have been obtained bycesarean, and that these animals could also be obtained after embryodelivery to sterile recipient mice (Okamoto, M. and Matsumoto, T. 1999.Exp. Anim. 48: 59-62), suggest to us that it has to be other mechanismsthan vaginal contamination that also influences the initial colonizationof the neonate, and that this mechanism has to begin before labor.

[0010] In this regard, to our knowledge, there is no publication or workdescribing the presence of lactic acid bacterial strains in normal humanamniotic fluid. Neither it has been reported that such non-pathogenicmicrobial strains could be beneficial for the gestating baby, andtherefore conditioning, just during the gestation, the initial microbialpopulations able to colonize the fetal gut.

[0011] In understanding the valuable properties that particular strainsof lactic acid bacteria may provide, there is a desire in the art foradditional lactic acid bacterial strains that are beneficial to the wellbeing of man and/or animal. Consequently, a problem of the prior art wasto provide rational methods for the selection of additional newbacterial strains and novel sources for the selection of them, thatallow the obtention of bacterial strains which exhibit individually ahigh number of beneficial properties for man and/or animals. The aboveproblem has been solved by providing novel microorganisms, namely lacticacid bacteria, belonging to the genus Lactobacillus.

[0012] These new strains have been obtained from different sources apartfrom feces, such as goat cheese and from human breast milk and amnioticfluid, and have been chosen by a method consisting in the ability ofthese strains to survive in breast milk and/or amniotic fluid, and bytheir ability to be transferred to breast milk and/or amniotic fluidafter oral intake.

[0013] This selection method ensures that the bacterial strains obtainedhave implicitly most of the characteristics attributed to a potentialprobiotic strain, namely good resistance to digestion process and theability of gut colonization, but also a more natural human origin,safety aspects, and the ability to colonize and regulate some humanniches other than the gut. Finally, the selected strains have also beentested not only for their adhesion capabilities but for having a highdegree of beneficial characteristics.

BRIEF DESCRIPTION OF THE FIGURES

[0014]FIG. 1. Screening method: Survival of bacteria into breast milkand amniotic fluid.

[0015] This figure illustrates the survival rate of the selectedprobiotic strains of this invention in breast milk and amniotic fluid.According to the selection method, the survival of the potentialprobiotic strains was measured using human breast milk (grey bars) andamniotic fluid (black bars). 10⁸ cfu of any candidate bacterial strainwere resuspended in 1 ml of MRS or breast milk or amniotic fluid, andincubated for 1 hour at 37° C. in anerobic conditions. Survival afterculture was assessed cultivating serial dilutions in MRS agar plates.The results are represented as the mean±SD of three independentexperiments. Those candidate bacteria that survive more than 75% in atleast one of human fluids were initially selected.

[0016]FIG. 2. Screening method: Transfer of bacteria to breast milk.

[0017] This figure illustrates the transfer of selected bacteria tobreast milk after oral intake. A) Labelling of the strains. Thosestrains that survive in breast milk and/or amniotic fluid weregenetically labeled using a PCR-detectable construction. Differentstrains are labeled with DNA fragments of different sizes (159 bp:F159;189 bp: F189; and 228 bp: F228). The PCR signal is only detectable inthe genetically labeled strains. The figure shows as example thelabeling of the bacterial strains CECT5711, CECT5713 and CECT5714. B)Transfer of selected bacteria to breast milk. The capability of thelabeled strains to be transferred to breast milk and/or amniotic fluidwas evaluated using pregnant mice as an animal model. Pregnant mice wereinoculated every two days with 10⁸ cfu/mice (in this figure we showedresults of L. salivarius CECT5713) for two weeks before labor. Presenceof bacteria in the milk was detected indirectly comparing the number ofbacteria PCR-detectable in the gut of neonates before and just afterlactating for the first time. Lane 1 in each panel corresponds to themolecular weight marker.

[0018]FIG. 3. RAPD profile of the selected bacteria.

[0019] This figure illustrates the RAPD profiles of the 5 selectedprobiotic strains of this invention using two different primers (Argdeiand OPL5). Lanes 1, 8 and 15 correspond to the molecular weight marker,lanes 7 and 14 are the negative controls.

[0020]FIG. 4. Comparison of the RAPD profile of the selected bacteriawith other bacteria of the same species.

[0021] This figure illustrates the differences in RAPD profiles observedbetween the selected probiotic strains of this invention and thoseobtained with other bacterial strains of the same species. Each specieis represented in one of the panels: L. coryniformis (A), L. salivarius(B), L. gasseri (C) and L. fermentum (D). The strains used are describedin Table III.

[0022]FIG. 5. Adhesion of probiotic strains to intestinal cells.

[0023] This figure illustrate the adhesión of probiotic strains tointestinal cells. The adhesion of the probiotic strains of thisinvention were assessed using Caco-2 (grey bars) or HT-29 (black bars)intestinal cell lines and compared to commercial probiotic strains.Twenty randomized fields were counted and the results expressed as themean of the number of bacteria attached to the cells per field±SD. Theadhesion capability of a probiotic strain to each intestinal cell linewas considered high if the number of attached bacteria was >250,moderate between 100 and 250, and slight >100.

[0024]FIG. 6. Survival of probiotics strains to digestion condition.

[0025] This figure illustrates the survival of probiotic strains todigestion conditions. The resistance of the probiotic strains of thisinvention to acidic (grey bars) and high bile salt content (black bars)was assessed in vitro by culture of bacteria in MRS pH 3.0 or 0.15% bilesalts for 90 minutes. The results are represented as the mean±SD ofthree independent experiments. The resistance of a probiotic strain wasconsidered high if the survival was >80%, moderate between 60% and 80%,and slight >60%.

[0026]FIG. 7. Generation time of probiotic strains.

[0027] This figure illustrates the generation time of probiotic strains.The time of generation of the probiotic strains of this invention wasassessed in vitro by cultivating bacteria in MRS 0.2% glucose for 120minutes. The results are represented in minutes and as the mean±SD ofthree independent experiments. The generation time of a probiotic strainwas considered rapid if the time was <60, moderate between 60 and 120,and slow >120 minutes.

[0028]FIG. 8. Fermentation capabilities of probiotic strains

[0029] This figure illustrates the ability of probiotic strains tofermentate complex carbohydrates. The fermentation capabilities of theprobiotic strains of this invention to use complex carbohydrates as anunique source of carbohydrates was assessed in vitro by cultivatingbacteria in MRS without glucose and supplemented with 2% of indicatedcarbohydrates for 24 and 48 hours. Reduction of the pH was assessedusing bromcresol purple. The results are represented as thefold-induction in absorbance after 24 hours compared with a controlculture without carbohydrate source (A) and the sum of all independentfold-induction values (B). The fermentation capability of a probioticstrain was considered high if the total value was >30, moderate between25 and 30, and slight <25.

[0030]FIG. 9. Resistance to antibiotic of probiotic strains.

[0031] This figure illustrates the resistance to antibiotic of probioticstrains. The resistance of antibiotic treatment of the probiotic strainsof this invention was assessed in vitro by an agar well diffusion assayin Müeller-Hinton plates for 24-48 hours. The diameter of the hallo ofinhibition determines the antibiotic effect. The results are representedas R (resistant) if the hallo has a diameter <12 mm, I (intermediate)from 12 to 15 mm, and S (sensible) if>15 mm. After that, a numericalvalue was assigned to each condition: R=3, I=2, and S=1. The resistancecapability of a probiotic strain was considered high if the total valuewas >17, moderate between 15 and 17, and slight <15.

[0032]FIG. 10. Acid production by the probiotic strains.

[0033] This figure illustrates the acid production by the probioticstrains. The production of acid (lactic, propionic, acetic and butyricacid) by the probiotic strains of this invention was assessed in vitroby the measurement of the pH in milk cultures for 24 (grey bars) and 48(black bars) hours. The production of acid by a probiotic strain wasconsidered high if the milk pH value after 48 hours was <4, moderatebetween 4 and 4.5, and slight >4.5.

[0034]FIG. 11. Production of bactericide metabolites by the probioticstrains.

[0035] This figure illustrates the production of antimicrobialmetabolites by the probiotic strains. The production of antimicrobialmetabolites by the probiotic strains of this invention was assessed invitro by an agar well diffusion assay in TSA plates cultured with S.typhimuriumi (black bars) or Escherichia coli (grey bars). The diameterof the hallo (in millimeters) of inhibition induced by the bacterialsupernatants determines the bactericide effect. The antimicrobialcapability of a probiotic strain was considered high if the hallowas >12, moderate between 8 and 12, and slight <8 for both pathogenicstrains.

[0036]FIG. 12. Inhibition of the adhesion of pathogenic bacteria.

[0037] This figure illustrates the inhibition of the adhesion ofpathogenic bacteria. The adhesion of the pathogenic strains E. coli(grey bars) and S. typhimurium (black bars) to Caco-2 cells was assessedin the presence of the probiotic strains of this invention and comparedto commercial probiotic strains. Ten randomized fields were counted andthe results expressed as the mean of the % of adhered gram-negativebacteria attached to the cells compared to the number of pathogenicbacteria adhered in absence of probiotics. The capability of a probioticstrain to inhibit pathogenic bacteria adherence was considered high ifthe % of both attached pathogenic bacteria was <25, moderate between 25and 75, and slight >75.

[0038]FIG. 13. Gut colonization by L. Salivarius CECT5713.

[0039] This figure illustrates the gut colonization by L. salivariusCECT5713. The number of fecal lactobacillus, bifidobacteria and coliformbacteria in mice supplemented daily for 14 days with 10⁸ cfu of L.salivarius CECT5713 was analyzed by bacterial platting. Fecal samples(200 mg aprox) were collected at day 0, 7 and 14 of probioticsupplementation and also one and two weeks (day 21 and 28) aftersupplementation was terminated. (*p<0.05; **p<0.01).

[0040]FIG. 14. Effect of L. Fermentum CECT5716 on Salmonella infection.

[0041] This figure illustrates the effect of L. fermentum CECT5716 onSalmonella infection. A) L. fermentum CECT5716 inhibits Salmonellatranslocation to the spleen. The number of Salmonella colonies wasmeasured in the spleens of mice treated with L. fermentum CECT5716 withor without vaccination with 108 inactivated cfu of Salnonella after 24hour of an oral challenge with 10¹⁰ cfu Salmonella. B) The same micewere used to measure the IgA content in feces.

[0042]FIG. 15. Effect of probiotic strains on cytokine expression.

[0043] This figure illustrates the effect of probiotic strains oncytokine expression. The TNF-α (A) ot IL-10 (B) cytokine production wasanalyzed in bone marrow derived macrophages stimulated with LPS and theindicated probiotic strain for 12 hours. Cytokine production wasdetected by an ELISA technique.

[0044]FIG. 16. Effect of probiotic strains on Ig G expression.

[0045] This figure illustrates the effect of probiotic strains on Ig Gexpression. The IgG production was analyzed in lymphocytes obtained fromthe spleen of Balb/c mice (6-8 weeks old) stimulated with LPS and theindicated probiotic strain for 6 days. Immunoglobulin production wasdetected by an ELISA technique from Bethyl.

SUMMARY OF THE INVENTION

[0046] The present invention provides, therefore, a method for theselection of probiotic microbial strains, comprising the followingsteps:

[0047] a. selecting for non-pathogenic strains which are capable ofsurviving in breast milk and/or amniotic fluid, and

[0048] b. selecting for non-pathogenic strains which are able to betransferred to breast milk and/or amniotic fluid after oral intake inhealthy individuals without colonizing other internal organs exceptmucousas.

[0049] In a further aspect, the invention provides new Lactobacillusstrains, which are:

[0050] CECT5711 (Lactobacillus coryniformis),

[0051] CECT5713 (Lactobacillus salivarius subsp. salivarius),

[0052] CECT5714: (Lactobacillus gasseri, formerly L. acidophilus),

[0053] CETC5715: (Lactobacillus gasseri), and

[0054] CECT5716: (Lactobacillus fermentum).

[0055] A further aspect of the invention relates to the use of mammalmilk and mammal amniotic fluid as a source to obtain non-pathogenicprobiotic bacteria.

[0056] Another aspect of the invention relates to compositions andproducts contaning at least one of the strains mentioned above.

[0057] Finally, a last aspect of the invention relates to the use of thestrains mentioned above or of any culture, composition or productcontaining them in the manufacture of a product for the therapeutic orprophylactic treament of human and animal diseases.

DETAILED DESCRIPTION OF THE INVENTION

[0058] As mentioned above, the present invention provides a method ofselection of new bacterial strains consisting in the ability of thesestrains to survive in breast milk and/or amniotic fluid, and by theirability to be transferred to breast milk and/or amniotic fluid afteroral intake, which ensures the special characteristics of the selectedstrains obtained with it. Thus, the main aspect of the present inventionis defined as a method for the selection of probiotic microbial strains,comprising the following steps:

[0059] a. selecting for non-pathogenic strains which are capable ofsurviving in breast milk and/or amniotic fluid, and

[0060] b. selecting for non-pathogenic strains which are able to betransferred to breast milk and/or amniotic fluid after oral intake inhealthy individuals without colonizing other internal organs exceptmucousas.

[0061] According to a preferred embodiment of the invention, both breastmilk and amniotic fluid are from human sources. The probiotic testedstrains in the method of the invention can be any probiotic bacteriaselected from, but not restricted to, the genera Lactobacillus,Lactococcus, Leuconostoc, Enterococcus, Streptococcus andBifidobacterium. These probiotic strains are preferably obtained frombreast milk, feces of breastfed babies or amniotic fluid, mostpreferably from human samples. Further details of the method of theinvention are given below in “Method and Examples”.

[0062] In a further aspect, the present invention provides any bacterialstrain selected by the method of the invention. Some of these newbacterial strains which exhibit a number of characteristics which renderthem beneficial to human health, and in particular in the prophylaxis ortreatment against digestive, infective, neuro-degenerative and otherimmune related diseases such as allergies or inflammatory diseases, havebeen deposited according to the Budapest Agreement at the CECT—ColecciónEspañola de Cultivos Tipo-, Valencia (Spain) on Jun. 11, 2002. Thesebacterial strains and their characteristics are:

[0063] CECT5711: (Lactobacillus coryniformis), said bacteria beingobtained from goat cheese, selected by the proposed process, andcharacterized by the RAPD profile showed in FIG. 3 and the featuresdescribed in Table I, II and VIII.

[0064] CECT5713: (Lactobacillus salivarius subsp. salivarius), saidbacteria being obtained from human breast-fed baby feces, selected bythe proposed process, and characterized by the RAPD profile showed inFIG. 3 and the features described in Table I, II and to VIII.

[0065] CECT5714: (Lactobacillus gasseri, formerly L. acidophilus), saidbacteria being obtained from human breast milk, detected also in humanamniotic fluid, selected by the proposed process, and characterized bythe RAPD profile showed in FIG. 3 and the features described in Table I,II and VIII.

[0066] CECT5715: (Lactobacillus gasseri), said bacteria being obtainedfrom human breast milk, detected also in human amniotic fluid, selectedby the proposed process, and characterized by the RAPD profile showed inFIG. 3 and the features described in Table I, II and VIII.

[0067] CECT5716: (Lactobacillus fermentum), said bacteria being obtainedfrom human breast milk, selected by the proposed process, andcharacterized by the RAPD profile showed in FIG. 3 and the featuresdescribed in Table I, II and VIII.

[0068] Moreover we have also confirmed that by applying the method ofthe invention, it is possible to select new specific strains as thementioned above. In this sense, we have compared genetic (RAPD profiles)and biochemical aspects such as fermentation capabilities (APIprofiles), enzymatic potential (APIZYM profiles) and antibioticresistance of the selected strains with other strains of the samespecies deposited in several culture collections (namely CECT, ATCC,LMG, NCFB, etc . . . ). Accordingly, we have stablished that these newstrains are different from those previously reported.

[0069] To our knowledge, this is the first time that an experimentallaboratory has observed that it is possible to obtain non-pathogenicbacterial microorganisms present in normal mammal breast milk oramniotic fluid, that exists a transfer of non-pathogenic bacterialstrains to breast milk and amniotic fluid after oral intake, and thatthese microbial organisms are not pathogenic strains that could act asprobiotic bacterial strains and thus, beneficially affect not only thesubject who ingest them but also to the fetus or breast feeding infant.For this reason, a further aspect of the present invention refers to theuse of the mammal breast milk and amniotic fluid as new sources ofbacterial microorganisms having the ability to be used as probioticstrains, the strains obtained from them, and the use thereof. The breastmilk and amniotic fluid are preferably human.

[0070] It is also an aspect of the invention comprising any of thebacterial strains of the invention together with at least anotherbacterial strain. In this regard, this invention refers to biologicalpure cultures of each of the strains, or mixtures among them or withother bacterial strains. Thus, said aspect of this invention is theproduction of different compositions comprising at least a strain or amixture of the strains of the invention. According to this aspect, theinvention provides a composition comprising at least one of thebacterial strains of the invention, i.e., one of the strains mentionedabove or any bacterial strain selected by the method of the invention,where the composition comprises preferably from 2 to 6 strains, morepreferably from 2 to 4 strains, most preferably from 2 to 3 strains, andwhere each of the strains is present in the composition in a proportionfrom 0.1% to 99.9%, preferably from 1% to 99%, more preferably from 10%to 90%. In a preferred embodiment, the composition comprises at leastone of the bacterial strains of the invention together with anotherstrain or mixture of strains where the mixture comprises preferably from2 to 6 strains, more preferably from 2 to 4 strains, most preferablyfrom 2 to 3 strains and where each of the strains is present in thecomposition in a proportion from 0.1% to 99.9%, preferably from 1% to99%, more preferably from 10% to 90%.

[0071] The compositions of the invention are preferably (no creo que seapreferible que sean asi, simplemente lo pueden ser) in a lyophilisedform, in a frost form or even dead.

[0072] In a further aspect, the present invention provides a compositionobtainable from the supernatant of a culture of a bacterial strain ofthe invention, or from any composition of the invention. In a preferredembodiment the composition is obtainable by extraction of a culture ofany of the bacterial strains of the invention or from a composition ofthe invention.

[0073] The supernatant of a culture of a Lactobacillus strain of thepresent invention may be used for preparing an administrable support.The supernatant may be used as such or may well be dried underconditions that do not destroy the metabolic compounds secreted orproduced by the microorganisms into the liquid medium, such as e.g.freeze drying. The present invention also refers to the use of enzymesobtained from these probiotic strains and their use in the production ofprotein hydrolysates or metabolites. The present invention also refersto compositions of the strains of this invention in a lyophilized form,freeze dried or inactivated (dead bacteria) by conventional methods.Thus yet in another aspect, the invention provides a product obtainablefrom the metabolic activity of any of the strains of the invention, of aculture of ay strain of the invention or of a composition according tothe present invention, wherein the product is preferably an enzyme.

[0074] A further aspect of the invention consists in a food productcomprising support material and at least one strain according to theinvention, a culture, a composition or a product according to thepresent invention. Preferably, the support material is a foodcomposition selected from milk, yoghourt, curd, cheese, fermented milks,milk based fermented products, meat based fermented products, fermentedcereals based products, milk based powders, cereal based powders, infantformulae, clinical nutrition formula, ice-creams, juices, flours, bread,cakes, sugar, candies or chewing-gums. In a preferred embodiment, themicrobial strain according to the instant invention is contained in thesupport material in an amount from about 10⁵ cfu/g to about 10¹² cfu/gsupport material, preferably from about 10⁶ cfu/g to about 10¹¹ cfu/gsupport material, more preferably from about 10⁶ cfu/g to about 10¹⁰cfu/g support material.

[0075] For the purpose of the present invention the abbreviation cfushall designate a “colony forming unit” that is defined as the number ofbacterial cells as revealed by microbiological counts on agar plates.

[0076] In another aspect the present invention provides pharmaceuticalcompositions comprising at least one strain according to the invention,a culture, a composition or a product according to the invention andpharmaceutically acceptable excipients. The required dosage amount inthe food or pharmaceutical composition described before will varyaccording to the nature of the disorder or the proposed use of thecomposition, whether used prophylactically or therapeutically and thetype of organism involved. For preparing a food composition according tothe present invention at least one of the Lactobacillus strains of thepresent invention is incorporated in a suitable support, in an amount offrom 10⁵ cfu/g to about 10¹⁴ cfu/g support material, preferably fromabout 10⁶ cfu/g to about 10¹³ cfu/g support material, more preferablyfrom about 10⁷ cfu/g to about 10¹² cfu/g support material. Thepharmaceutical preparations can be prepared in forms of tablets,capsules, liquid bacterial suspensions, dried oral supplements, wet oralsupplements, dry tube feeding or a wet tube feeding.

[0077] Nevertheless, the activity of the new microorganisms in theindividual is naturally dose dependent. That is, the higher the numberof the novel microorganisms that are incorporated by means of ingestionor administration of the above food material or the pharmaceuticalcomposition, the higher protective and/or therapeutic activity of themicroorganisms. Since the microorganisms of this invention are notdetrimental to man and animals and have eventually been isolated frombaby feces, food or human breast milk or amniotic fluid, a high amountthereof may be incorporated so that essentially a high proportion of theindividual's mucosa will be colonized by the novel microorganisms.

[0078] Preferably, the subject in need of treatment is selected from thegroup consisting of individuals who suffer the disorder or having riskto suffer the selected disorder, namely infection, allergy,inflammation, etc. However, it will be recognized that the presenttreatments are suitably employed in prophylaxis of those disorders inany subject.

[0079] Moreover, due to the ability of the selected strains to betransferred to and survive in breast milk and/or amniotic fluid, thesubjects in need of treatment could not only be those who intakedirectly the selected strains but also the fetus or breast feedingbabies.

[0080] Preferably the probiotic, or the probiotic-containingcomposition, is directed to the oral, gastric or to the intestinalmucosal surface; however, it could also be directed to naso-pharingeal,respiratory, genitourinary or glandular mucosa, and it could beadministered to human and animals by an oral, rectal, topical, urethralor vaginal route.

[0081] Further, the probiotics of the present invention may be used inconjunction with other treatments, to enhance or assist in theirefficacy.

[0082] Many people have a disturbed intestinal microflora, that is, thebalance between useful and harmful intestinal bacteria is disturbed. Anumber of factors, among others stress, the presence of bile salts, andspecially diet, influence the bacterial flora. In these situations thefermentation process could be disturbed and the number of usefulbacteria be reduced, the consequence would be that the colon mucosawithers away and ceases to function at the same time as the potentiallymalignant bacteria rapidly grow in number. For this reason, one aspectof this invention is the use of probiotics as prophylactic ortherapeutic treatment of chronic or acute infection, or of undesirablemicrobial colonization, of a mucosal surface, comprising theadministration of an effective amount of a probiotic, or aprobiotic-containing composition, to a subject in need thereof.

[0083] The compositions of the present invention can also be usedeffectively in the treatment of acute and chronic viral infections. Inparticular, the treatment of chronic Epstein-Barr virus, cytomegalovirusand other herpes-type virus infection, which are ubiquitous in thepopulation and are associated with a decrease on the immune survillance.

[0084] Another embodiment of the invention is the use of the probioticbacteria of this invention for the prophylactic or therapeutic treatmentof diarrhea, independently whether this disorder is due to the presenceof a parasitic infestation and/or bacterial or viral infection, thetreatment with antibiotics or quimio- or radio-therapy or to dietary orphysical complications.

[0085] The present invention also relates to the use of the statedprobiotics for the prevention and treatment of temporarily reducedimmune activity levels and normalizing immune activity levels that aredepressed in comparison with what may be considered normal, such as thatproduced in aging or in healthy individuals who are subject to intenseexertion or in general to a great physiological strain.

[0086] Moreover, through modulation of the immune response and thebalance between Th1 and Th2 cytokines, the probiotics of the inventioncould also be used for the prophylactic or therapeutic treatment ofallergy and disorders related with the development of tolerance againstingested proteins.

[0087] Another embodiment of the invention is the use of the probioticsof this invention for the prophylactic or therapeutic treatment ofchronic inflammatory disorders such as, but not restricted to,psoriasis, sarcoidosis, atherosclerosis, inflammatory bowel disease, dueto the ability of some of the probiotic strains to reduce the productionof pro-inflammatory cytokines by activated macrophages.

[0088] The present invention also relates to the use of the strainsstated in this invention for the prophylactic or therapeutic treatmentof some cancer types. This use of the strains is based on the describedeffects of some lactic acid bacteria counteracting cancer due to theireffects in the inhibition of carcinogenic toxins in the intestines suchas nitrosamines but also for the effect of this probiotics in themodulation of the natural immune defense.

[0089] Finally, the present invention also refers to the use of theseprobiotic strains for the prophylactic or therapeutic treatment ofneuro-degenerative diseases due to the hypocholesterolemic and themodulation of the oxidative stress effect of some strains of probiotics.Both situations have been related as risk factors for the development ofneuro-degeneratives diseases such as Parkinson or Alzheimer. Moreover,it has also been described that commensal bacteria are able to deaminateL-tryptophan producing indole-3-propionic acid which is a potentneuroprotective agent.

[0090] We have also tested the potential of the strains selected by theselection method of the present invention, analyzing the probioticproperties of the selected strains using conventional criteria. In thissense we have studied the following aspects: a) Acid and bile stability,because the bacteria are mainly ingested and must pass through theacidic environment of the stomach as well as the bile-containing smallintestine, and they must be able to survive in these conditions; b)Adherence to intestinal mucosa, because this property permits thebacteria to colonize and become established in the gastrointestinaltract; c) Fermentative and high proliferative capabilities, to enhancethe establishment in the mucosa; d) Resistance to antibiotics, becauseit could be necessary for some indications; e) Reduction of pH (lacticacid production) and production of antimicrobial metabolites by thestrains of the invention, since it can help them to form a protectivebarrier to pathogens within the gastrointestinal tract; f)Immunomodulatory capabilities.

[0091] The following methods and examples illustrate the invention.

METHODS AND EXAMPLES Example 1 New Method of Selection of ProbioticStrains

[0092] We have developed a novel method of selection of new bacterialstrains consisting in the ability of these strains to survive in breastmilk and/or amniotic fluid, and by their ability to be transferred tobreast milk and/or amniotic fluid after oral intake. The rationale ofthis novel method described in the present invention is that ensuresspecial characteristics of the selected strains obtained with it, sincethe bacterial strains obtained have implicitly most of thecharacteristics attributed to a potential probiotic strain, namely goodresistance to digestion process and the ability of gut colonization, butalso a more natural human origin, safety aspects, and the ability tocolonize and regulate some human niches other than the gut. Moreover,these new strains have been obtained from different sources apart fromfeces, such as goat cheese and from human breast milk and amnioticfluid.

Example 1a: Resistance to Human Fluids

[0093] Colonies isolated from different sources were checked by theirability to survive in human breast milk and also in human amnioticfluid. To analyze the survival rate of the probiotic strains of thisinvention, 10⁸ cfu of each bacteria were cultured in 1 ml of humanbreast milk or human amniotic fluid for 60 minutes in anaerobicconditions at 37° C. The survival was calculated by MRS agar plating ofserial dilutions and compared to the number of colonies obtained incontrol conditions (MRS broth pH 6.2). Plates were cultured 16-18 hoursat 37° C. in anaerobic conditions. The experiment was repeated threetimes. Strains were considered resistant when the survival at least inone of the human fluids was higher than 75% compared with the controlconditions (FIG. 1).

Example 1b: Transfer to Human Fluids

[0094] The second criteria in the selection process described in thepresent invention is that bacteria should be able to be transferred tobreast milk and/or amniotic fluid after oral intake. In order to testthis capability, the putative strains were genetically labeled, asdescribed latter, and orally administered to pregnant mice as animalmodel. Transfer of bacteria was analyzed by PCR screening of thecolonies obtained from the amniotic fluid and from the gut of breastfedmice.

[0095] Labeling of Bacteria:

[0096] Three primer couples were employed to obtain three different PCRfragments (F159: 159 bp, F189: 189 pb, and F228: 228 bp, respectively).The three fragments included the junction between the 35S rRNA promoterof the Cauliflower Mosaic Virus (CaMV) and the5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene fromAgrobacterium tumefaciens. The primers were designed from the artificialsequence present in Roundup Ready soya (EMBL accession number:AX033493). BamHI sites were added to the 5′-tails of all the primers tofacilitate the cloning of the PCR fragments. Once obtained, the PCRproducts were purified using the QIAquick PCR purification kit (Qiagen),digested with BamHI, and ligated into pTG262, a plasmid that confersresistance to Chloramphenicol (Cm). Subsequently, these plasmids wereindividually introduced into the selected strains by electroporationfollowing conventional protocols. Identity of the transformants wasconfirmed by PCR (FIG. 2A).

[0097] Transfer of Bacteria:

[0098] Four pregnant Balb/c mice were orally inoculated with 10⁸ cfu ofgenetically-labelled strains vehiculated in 200 μl of milk every twodays from two weeks before labor. Just before labor, amniotic fluid wasaseptically collected from two of the mice, and cultured on MRS agarplates. The other two pregnant mice finalized gestation. The transfer ofthe genetically labeled bacteria to breast milk was analyzed bycomparison of the bacteria isolated from the neonate's gut just beforeand after first lactation. All the plates were incubated for 24 h at 37°C. under anaerobic conditions. For each sample obtained, 52 colonieswere randomly selected among those that grew on the MRS plates andsubcultured on Cm-MRS plates. Finally, to detect the genetically-labeledcolonies among the Cm-resistant colonies, PCR analyses were performedusing DNA from the Cm-resistant colonies as template (FIG. 2B). Transferwas considered positive when at least two PCR-positive colonies could bedetected in at least one of the samples.

[0099] The bacterial strains selected by the method described in thepresent invention were further tested in order to establish theirsingularity and their probiotic properties as described in the examples3 and 4.

Example 1c Isolation of Lactic Acid Bacteria

[0100] The bacterial strains that have been submitted to the selectionmethod described in the present invention have been obtained fromdifferent sources apart from feces, such as goat cheese and from humanbreast milk and amniotic fluid. Tills isolation process have beenperformed as described above:

[0101] Isolation from Human Breast Milk:

[0102] Two milliliter samples of human breast milk was collectedaseptically from a 35 year-old woman (15 days after delivery). In orderto isolate bacterial strains from this sample, serial dilutions of 0.1ml in peptone water were plated on MRS (pH 6.2), MRS (pH 5.5), APT, RCM,LM17, GM17 and Elliker agar plates at 37° C. in both aerobic andanaerobic conditions for 24-48 hours. From about 740 colonies in total,74 colonies (10%) that included at least two colonies of the appreciateddifferent morphologies were selected and further cultured in MRS agar at37° C. in anaerobic conditions and tested according to the proposedmethod. Two of the colonies obtained from this sample were able tofulfill the defined criteria.

[0103] The selected breast milk-derived Lactobacillus gasseri CECT5714and Lactobacillus fermentum CECT5716 were originally isolated from theMRS (pH 6.2) agar plates cultured in aerobic conditions, whereas theLactobacillus gasseri CECT5715 was isolated from the APT agar platescultured in anaerobic conditions.

[0104] Isolation from Human Amniotic Fluid:

[0105] Isolation of bacterial strains from human amniotic fluid wasperformed by dilution of 2 ml of human amniotic fluid collectedaseptically by the clinical staff during labor from two volunteers.Serial dilutions of 0.1 ml in peptone water were plated on MRS (pH 6.2),MRS (pH 5.5), APT, RCM, LM17, GM17 and Elliker agar plates at 37° C. inboth aerobic and anaerobic conditions for 24-48 hours. From about 400colonies in total, 40 colonies (10%) that included at least two coloniesof the appreciated different morphologies were selected and furthercultured in MRS agar at 37° C. in anaerobic conditions and testedaccording to the proposed method.

[0106] The two strains selected from this source were identical to thosepreviously selected from human breast milk, namely Lactobacillus gasseriCECT5714 and CECT5715.

[0107] Isolation from Food Products (Goat Cheese):

[0108] Isolation of bacterial strains from food products was carried outby homogeneization in peptone water of 20 g of a central part of thefood product collected aseptically. 0.1 ml of serial dilutions wereplated in MRS agar (Oxoid) plates and RCM agar (Oxoid) plates andcultured in both aerobic and anaerobic conditions at 32° C. for 48hours. From more than 500 colonies in total, 5 colonies of eachcondition were selected and further cultured in MRS agar at 37° C. inanaerobic conditions. These colonies were tested according to theproposed method. Only one of the colonies was able to fulfill thedefined criteria. The selected cheese-derived colony Lactobacilluscoryniformis CECT5711 was originally isolated from MRS agar platescultured in aerobic conditions.

[0109] Isolation from Human Breast-Fed Baby Feces:

[0110] Isolation of bacterial strains from human feces was performed byhomogenization of 2 g of feces collected aseptically from threeindependent babies (15-45 day old) in peptone water. 0.1 ml of serialdilutions were plated on MRS (pH 6.2), MRS (pH 5.5), APT, RCM, LM17,GM17 and Elliker agar plates at 37° C. in both aerobic and anaerobicconditions for 24-48 hours. From about 670 colonies in total, 67colonies (10%) that included at least two colonies of the appreciateddifferent morphologies were selected and further cultured in MRS agar at37° C. in anaerobic conditions and tested according to the proposedmethod. Only one of the colonies was able to fulfill the definedcriteria.

[0111] The selected baby feces-derived Lactobacillus salivarius subsp.salivarius CECT5713 was originally isolated from the MRS (pH 6.2) agarplates cultured in aerobic conditions.

Example 2 Physiological and Genetic Characterization

[0112] The phenotype of each selected bacterial strain grown on MRSmedia (agar or broth) at 37° C. in anaerobic conditions was as describedin Table I: TABLE I Phenotypic characteristics of the differentprobiotic strains of the invention. The phenotypic characteristics ofthe different probiotic strains of the invention were compared to thatobserved in known commercial probiotic strains (Lactobacillus rhamnosusLGG from Valio, Lactobacillus johnsonii La1 from Nestlé andLactobacillus casei immunitas from Danone). TEST CECT5711 CECT5713CECT5714 CECT5715 CECT5716 LGG LA1 LC Origen cheese feces breast milkbreast milk breast milk feces feces feces Gram + + + + + + + + catalase− − − − − − − oxidase − − − − − − − morphology rod rod rod rod rod rodrod small rod size (μm) 1 × 1.5 − 4 0.9 × 15 − 3 0.9 × 2 − 4 1 × 2 − 101 × 1.5 − 3 1 × 2 − 4 0.9 × 1.5 − 3.5 0.9 × 15 − 2 motility nonmotilenonmotile nonmotile nonmotile nonmotile nonmolile nonmotile nonmotileagregation single/pairs single/pairs single/pairs single/pairssingle/pairs long chains single/pairs single/pairs

[0113] For the identification of the selected probiotic strains afermentation API 50CH (BioMerieux) analysis at 37° C. in anaerobicconditions for 24 and 48 hours was carried out following the specifiedinstructions indicated by the manufacturer. The results after 24 hoursof culture are summarized in Table II. A positive fermentable substrateis that with a value higher than 3.

[0114] The selected bacterial strains were taxonomically classifiedaccording to their SDS-PAGE ID protein profiling and 16S rDNA sequenceby BCCMILMG (Belgium) and/or NIZO Food Research (The Netherlands),respectively. The results obtained from these tests lead to thetaxonomical classification of the bacterial strains as indicated above.With this classification the bacterial strains of the invention weredeposited according to the Budapest Agreement at the CECT—ColecciónEspañola (le Clultivos Tipo-, Valencia (Spain) on June 11^(Th) 2002 andwith the following accession numbers:

[0115]Lactobacillus coryniformis: CECT5711

[0116]Lactobacillus salivarius subsp. salivarius: CECT5713

[0117]Lactobacillus acidophilus. CECT5714

[0118]Lactobacillus gasseri: CECT5715

[0119]Lactobacillus fermentum: CECT5716

Example 3 Singularity of the Selected Strains

[0120] Although the analysis of the SDS-PAGE ID protein profiling and16S rDNA sequence performed in example 2 are suitable methods to definebacterial species, they have not enough specifity to discriminatebetween different strains of the same bacterial species. For thisreason, RAPD-PCR analysis of the strains was performed using twodifferent lactobacilli specific primers (ArgDei and OPL5). For theRandomly Amplified Polymorphic DNA (RAPD)-PCR analysis, genomic DNA wasisolated from 10 ml of overnight MRS cultures using the DNeasy tissuekit (Qiagen) and following the protocol recommended by the supplier forisolation of genomic DNA from Gram-positive bacteria. Total DNA was usedin subsequent PCR amplifications carried out in a Techne DNA ThermalCycler. PCR amplifications were performed using either primer OPL5(5′-ACGCAGGCAC-3′), or ArgDei (5′-ACCYTRGAAGGYGGYGATGTB-3′). Five μl ofthe PCR mixtures were analyzed on a 1.2% (wt/vol) agarose (Sigma) gelwith ethidium bromide staining. A 100-bp ladder (Invitrogen) was used asa molecular weight standard. Gels were run for approximately 1 h at 100V, and the DNA was visualized and analyzed in a gel documentation system(Gel Doc 2000, Bio-Rad), using the Diversity Database software package(Bio-Rad).The results are showed in FIG. 3.

[0121] Moreover, in order to test the singularity of the bacterialstrains selected with this new process and compare them with strainsobtained by other selection criteria but that had been previouslyassigned to the same species. These probiotic strains were obtained fromseveral culture collections such as CECT, ATCC, LMG or DSM and describedin Table III. TABLE III Probiotic strains used for testing thesingularity of the probiotics included in this invention. Lactobacilluscoryniformis DSM 20005: Lactobacillus coryniformis subsp. torquens DSM20007: Lactobacilus coryniformis subsp. coryniformis CECT 982:Lactobadilus coryniformis subsp. coryniformis CECT 4129: Lactobadiluscoryniformis subsp. torquens Lactobacillus fermentum LMG 8900:Lactobacillus fermentum = ATCC 11976 LMG 17551: Lactobacilus fermentum =ATCC 23271 CECT 285: Lactobacilus fermentum = ATCC 9338 CECT 4007:Lactobacilus fermentum = ATCC 14931 Lactobacillus gasseri LMG 11413:Lactobacillus gasseri LMG 13047: Lactobacillus gasseri = ATCC 19992 LMG13134: Lactobacillus gasseri = ATCC 9857 LMG 18176: Lactobacillusgasseri LMG 18194: Lactobacilus gasseri CECT 4479: Lactobacilus gasseriLactobacillus salivarius DSM 20492: Lactobacillus salivarius CECT 4062:Lactobacillus salivarius CECT 4063: Lactobacillus salivarius

[0122] All the selected strains included in this invention were comparedwith the strains described in Table III regarding their RAPD-PCRprofiles using two different primers, results of this analysis (FIG. 4)shown that the selected strains included in this invention are differentto those previously described. Moreover, we extended our results andcompared not only genetic characteristics but also biochemical aspectsof the selected strains with those strains described in Table III. Inthis sense, we performed API analyses (BioMerieux) (Table IV), APIZYManalyses (BioMerieux) (Table V) and antibiotic resistance as describedin example 5 g (Table VI). The activities that differ to that observedwith the strains of this invention has been indicated in grey.

[0123] Probiotic strains used were described in Table III. Theactivities that differ to that observed with the strains of thisinvention has been indicated in grey. TABLE VI Comparison of antibioticresistances of the selected bacteria using the method described in thisinvention with other bacteria of the same species.

[0124] Probiotic strains used were described in Table III. Theantibiotics used and the methodology is described in example 4e.R=resistant, I=intermediate, S=sensible. The activities that differ tothat observed with the strains of this invention has been indicated ingrey.

Example 4 Probiotic Characteristics of the Strains

[0125] We also analyzed the suitability of the probiotic selectionprocess included in this invention regarding its ability to selectbacterial strains with desirable probiotic characteristics. In order toevaluate this, the selected strains were analyzed for a high number ofdifferent characteristics that could enhance their capabilities to actas a probiotic strains. Moreover, we assigned arbitrarialy (asindicated) a numerical value to the results obtained in each test inorder to compare the probiotic strain included in this invention tothose obtained by other selection criteria. The results obtained aresummarized in Table VIII at the end of this example and described in thefollowing sub-examples and compared with some commercial strains.

Example 4a Adhesion Analysis to Caco-2 and HT-29

[0126] Culture of Caco-2 and HT-29 Cells

[0127] For the adhesion and inhibition assays, the cell lines Caco-2(ATCC HTB-37) and HT-29 (ATCC HTB-38) were utilized as a model of theintestine cells. Both cell lines presented features characteristic forintestinal cells such as polarization, expression of intestinal enzymes,and production of particular structural polypeptides and mucins.

[0128] The cells were grown in plastic flasks (75 cm², Nunc) in DMEM(PAA laboratories) as culture medium supplemented with 10% inactivatedFCS (Fetal Calf Serum, PAA laboratories), non essential aminoacids, 100U/ml penicilline/streptomycine, 1 μg/ml amphoterine. Cell culture wasperformed at 37° C. in an atmosphere comprising 95% air and 5% CO₂.Media was changed on a two daily basis and the cells were splitted everyweek.

[0129] For the adhesion assays the cells were splitted to 35 mm plasticdishes (Nunc) and cultured in similar conditions but without antibioticsafter confluence. Adhesion assays were performed 10-14 dayspost-confluence.

[0130] Culture of Bacteria

[0131] Probiotic Strains:

[0132] The probiotic strains of this invention were cultured in MRSbroth (pH 6.2) in anaerobic conditions for 16-18 hours at 37° C. afterinoculation of a 0.1% (v/v) from the glycerol stock. In this conditions,the concentration of the culture was 1-2×10⁹ cfu/ml, as observed byplating on MRS agar.

[0133] Gram-Negative Strains:

[0134]Escherichia coli 0157:H7 (non-pathogenic) (CECT4972), E. coli0157:H7 (entero-pathogenic) (CECT4783), E. coli 0157:H7(entero-pathogenic) (CECT4782), Salinonella cholerasuis typhi (CECT409)and S. cholerasuis typhimurium (CECT443) were all obtained from theCECT-Colección Española de Cultivos Tipo-. All gram negatives strainswere cultured in TSB broth (AES Laboratoire) in anaerobic conditions for16-18 hours at 37° C. after inoculation of a 0.1% (v/v) from theglycerol stock. At this conditions, the concentration of the culture was1-2×10⁹ cfu/ml, as observed by plating on TSA agar (AES Laboratoire).

[0135] Adhesion Analysis

[0136] Caco-2 and HT-29 intestinal cell lines were cultured in 35 mmplastic dishes in 2 ml medium without antibiotics to confluence. 10-14days post-confluence 1 ml of media was replaced with 1 ml of asuspension of 10⁸ bacteria in DMEM. The cultures were incubated 1 hourat 37° C. After that, cells were washed twice with PBS and fixed withice-cold 70% methanol for 30 minutes. Plates were air dried and Gramstained. The attached bacteria were visualized using an optical Axiovert200 (Zeiss) microscope at 1000× magnification in oil-immersion. Twentyrandomized fields were counted and the results expressed as the mean ofthe number of bacteria attached to the cells per field±SD. Thecapability of a probiotic strain was considered high if the number ofattached bacteria was >250, moderate between 100 and 250, andslight >100 (FIG. 5)

Example 4b Resistance to Acid and Bile Salts

[0137] To analyze the resistance of the probiotic strains of thisinvention to acidic and high bile salt content, conditions that thesebacteria will encounter during the digestive transit, bacteria werecultured in MRS broth media either at pH 3.0 or in MRS broth at pH 6.2supplemented withO.15% bile salts (Sigma) for 90 minutes. The survivalrate was calculated by MRS agar plating of serial dilutions and comparedto the number of colonies obtained in control conditions (MRS broth pH6.2). Plates were cultured 16-18 hours at 37° C. in anaerobicconditions. The experiment was repeated three times. Resistance wasconsidered high when the survival was >80%, moderate 80% to 60%, slight<60% compared with the control conditions (FIG. 6).

Example 4c Time of Generation

[0138] The time of generation, meaning the time that a bacterial culturerequires to duplicate the concentration of bacteria, is an importantcharacteristic for a probiotic bacteria. It is important from anindustrial point of view (production of a higher amount of biomass inthe same amount of time) and from a probiotic point of view (highercolonization of the gut). In order to consider both aspects we haveanalyzed the generation time of the probiotic strains of the inventionin a rich media (industrial point of view) and in a poor media(probiotic point of view).

[0139] The probiotic strains of this invention were grown in MRS broth(pH 6.2) with 2% (rich media) or 0.2% (poor media) glucose for 0, 1, 2,4 and 6 hours at 37° C. in anaerobic conditions and the concentration ofbacteria was determined by plating serial dilutions in MRS agar platesand incubation of the plates for 16-18 hours at 37° C. in anaerobicconditions (FIG. 7). The generation time was calculated as the time inminutes necessary in order to duplicate the number of colonies at theinitial time.

Example 4d Fermentation Capabilities

[0140] The capacity of a bacterial strain to metabolize complexcarbohydrates (soluble and non-soluble fibers) ensures that theseprobiotic strains could use them as a carbon source in the colon, andthus enhance the efficiency of colonization. For this reason, we havetested the capability of the probiotic strains of this invention to useseveral non-digestible fibers as an unique source of carbohydrates.

[0141] To assay the capability to the probiotic bacterial strains tometabolize fiber we cultured, a liquid culture was carried out in MRSbroth media without glucose and supplemented with a 2% of each fiber in96 well flat-bottomed plastic dishes (Nunc) for 24 and 48 hours at 37°C. in anaerobic conditions. The fermentation process was controlled bypH decrease in the media and determined by a colorimetric approach using0.3% phenol red as indicator and measuring the absorbance at 540 nm.

[0142] The fibers used were: α-celulose (raw cellulose, Campi y Jove),Actilight (fructo-oligosaccharide, Beghin-Meiji), Ficao (cocoa fiber,Natra), Fructafit (Inulin, Sensus), Lactose (Bordulo), Pectine (YM100,Genu), Raftiline (Inulin oligofructose, Orafti), Raftilose (Inulinoligofructose, Orafti), and Vitacel (purified cellulose, Campi y Jove).

[0143] The fermentation capability (defined as fold-induction of the pHreduction compared to the control without fiber) was calculated. Resultsshowed in FIG. 8 represent the individual values for each fiber (panelA) and the sum of all these individual values for each selected strain(Panel B). Fermentation capability was considered high when the sum ofindividual values was >30, moderate 30 to 25, slight <25.

Example 4e Resistance to Antibiotics

[0144] The use of antibiotics leads to a reduction of the comensal gutmicroflora which sometimes relates to diarrhea and other gut disorders.Moreover, this reduction in the amount of gut bacteria could be theconsequence of opportunistic pathogenic bacteria and viruses to infectthe host. The use of antibiotics to block the infection does not resolvethis disorder but complicates it. In other situations like intestinalinflammation where probiotics could exert a beneficial role, thispotential effect is sometimes limited by the simultaneous therapy withantibiotics. For these reasons, the selection of potential probioticstrains able to resist common antibiotics would be an improvement in theart.

[0145] To analyze the resistance of the probiotic strains of thisinvention an agar well diffusion assay was used. Müeller-Hinton agarplates containing 10⁶ cfu/ml of each probiotic strain were prepared.Then, antibiotic commercial discs corresponding to the indicatedconcentrations were added to the wells and allowed to diffuse into theagar during a preincubation period of 10 minutes at room temperature,followed by anaerobic incubation of the plates at 37° C. for 16-18hours. Diameter of inhibition halos was measured and the resistancedegree of the bacteria to each antibiotic was graded as R (resistant), I(intermediate) or S (sensible) according to the described sensibility oflactobacilli to this antibiotics (Table VII). After that, a numericalvalue was assigned to each condition: R=3, I=2, and S=1. Ten differentantibiotics were tested and the numerical values were added up to get anoverall value. The resistance capability of a probiotic strain wasconsidered high if the total value was >17, moderate between 15 and 17,and slight <15.

[0146] The antibiotics and concentrations used were: Erythromycin 15 mg(E 15), Penicillin 10 μg (P 10), Ciprofloxacin 5 μg (CiP 5),Chloramphenicol 30 μg (C 30), Nalidixic 30 μg Na 30), Amoxicilin 10 μg(AM 10), Tetracycline 30 μg (Te 10), Vancomicin 30 μg (Va 30),Cephoxithin 30 μg (Fox 30), and Cephalothin 30 μg (CF 30) (FIG. 9).TABLE VII Resistance of the selected strains to antibiotics. TESTCECT5711 CECT5713 CECT5714 CECT5715 CECT5716 LGG LA1 LC E 15 S S S S S SR R P 10 S S S S S S S S CiP 5 R I R R R I R R C 30 S S S S S S S S Na30 R R R R R R R R AM 10 S S S S S S S S Te 30 I S S S S S S S Va 30 R RI I R R S S Fox 30 R S R R R R R R CF 30 S S I I S I I I Total 19 15 1618 19 17 18 18

Example 4f Production of Metabolic Acids

[0147] The production of metabolic acids by probiotic bacteria, namelylactic, acetic, propionic and butyric acid, and the subsequent reductionof the pH in feces has been extensively associated with a beneficialeffect of these bacteria due to a reduction in the growth and infectivecapabilities of opportunistic pathogenic microorganisms. Moreover, someof these acids, specially butyric acid, are rapidly absorbed and used bythe intestinal cells as an energy source. In this sense, reduced pH infeces of the breast feeding infants has been associated with the reducedrisk of gut disorders compared with formula feeding babies.

[0148] Acid producing capacity of the probiotic strains of the inventionwas observed by measurement of the pH reduction during milkfermentation. Five ml of skimmed milk were inoculated with 10⁸ cfu ofeach bacterial strain and fermented for 24 (grey bars) and 48 (blackbars) hours at 37° C. in anaerobic conditions and the pH was measuredusing a CyberScan 510 pHmeter (VWR). The production of acid by aprobiotic strain was considered high if the milk pH value after 48 hourswas <4.5, moderate between 4.5 and 5.5, and slight >5.5 (FIG. 10).

Example 4g Production of Antimicrobial Metabolites

[0149] It has been suggested that the main benefficial effect ofprobiotics is the control of the balance between useful and harmfulintestinal bacteria is the gut. When the number of useful bacteria isreduced, opportunistic bacteria could over-grow and disturb thewell-being of the host or even induce an infection. Most bacterialorganisms have adquired characteristics or mechanisms that reduce thegrowth capabilities of other microorganisms that cohabitate with themand thus, enabling their selective growth. As stated in example 4f, thereduction of pH through acid production by lactic acid bacteria is oneof such mechanisms. Moreover, some lactic bacteria also producebioactive peptides components and other metabolites that selectiveinhibit the growth of other bacteria, yeast or fungi. This is the caseof bacteriocins such as pediocin.

[0150] The probiotic strains of this invention were assessed for theircapability to produce antimicrobial metabolites using an agar welldiffusion assay. MRS agar plates containing 10⁶ cfu/ml of differentpathogenic bacteria strain (Salmonella typhmurium and Escherichia coli)were prepared. Wells, with a diameter of 5 mm, where then cut in theagar using a sterile cork-borer. Then, 50 μl of a 2 fold concentratesupernatant of each probiotic strain culture were added to the wells andallowed to diffuse into the agar during a 2 hours preincubation periodat 4° C., followed by aerobic incubation of the plates at 37° C. for16-18 hours. The antimicrobial activity of each supernatant wasconsidered high if the diameter of the inhibition hallo for bothpathogenic bacteria strains was >12, moderate between 8 and 12, andslight <8 (FIG. 11)

[0151] Moreover, it was also tested if the antimicrobial activity of thesupernatants was due to a antimicrobial substance or to the productionof metabolic acids. In this sense, the inhibitory effect of a dilutionof each metabolic acid (acetic, lactic, propionic and butyric) at pH 4.5was assayed. None of these situations inhibited the growth of Salmonellaor E. coli in these conditions (data not shown). It was also tested theantimicrobial capabilities of supernatants obtained from bacterialcultures using glucose or lactose as a carbohydrate source. In thesecircumstances, those bacterial strains that do not ferment lactose (L.rhamnosus GG and L. acidophilis CECT5714) did not showed antimicrobialactivity in the lactose-containing culture whereas they showed thisactivity it in the glucose culture (data not shown).

Example 4h Inhibition of Pathogen Adhesion to Caco-2

[0152] Caco-2 intestinal cell lines were cultured in 35 mm plasticdishes in 2 ml complete medium without antibiotics to confluence. 10-14days post-confluence 1 ml of media was replaced with 1 ml of asuspension of 10⁸ probiotic bacteria in DMEM. The cultures wereincubated 1 hour at 37° C. After that, 1 ml of a suspension of 10⁸pathogenic bacteria (E. coli or S. typhimurium) in DMEM was added to thecultures and incubated 1 hour more at 37° C. The cells were washed twicewith PBS and fixed with ice-cold 70% methanol for 30 minutes. Plateswere air dried and Gram stained. The attached bacteria were visualizedusing an optical Axiovert 200 (Zeiss) microscope at 1000× magnificationin oil-immersion. The number of gram-negative bacteria in 10 randomizedfields were counted and the results expressed as the mean of % ofpathogenic bacteria attached to the cells compared to control cultureswithout probiotic strains. The capability to inhibit the adhesion ofpathogenic bacteria to intestinal cells of a probiotic strain wasconsidered high if the % of both strains of Gram-negative attachedbacteria as compared with the control was <25%, moderate between 25% and75%, and slight >75% (FIG. 12).

[0153] All the results obtained in the example 4 are summarized in TableVIII. Each test was performed as indicated in the correspondingsub-example and described in this document. The categories in each testwere assigned as indicated. a) Example 4a; number of bacteria attachedper field; high >250, moderate: 100 to 250, slight <100. b) Example 4b;% of survival compared to control conditions; high >80%, moderate: 80%to 60%, slight <60%. c) Example 4c; minutes necessary to duplicate deinitial population; rapid <60, moderate: 60 to 120, slow >120. d)Example 2; number of fermentable substrates; high >18, moderate, 12 to18, slight <12. e) Example 4d; Accumulated fold-reduction of the totalfermentable substrates compared with the control; high >30, moderate: 25to 30, slight <25. f) Example 4e, Accumulated resistance to eachantibiotic (resistant=3, intermediate=2, sensible=1); high >17,moderate: 15 to 17, slight <15. g) Example 4f; pH value of milk after 48hours culture; high <4, moderate: 4 to 4.5, slight >4.5. h) Example 4g;mm of inhibition hallo high >12, moderate: 12 to 8, slight <8, (*) onlyin presence of glucose but not lactose. i) Example 4h; % of adhesion;high <25, moderate: 25 to 75, slight <75. The global probioticcapability was calculated by the sum of all tests (high=3, moderate=2,slight=1). TABLE VII Potency to act as a probiotic of the differentstrains of the invention. The different capabilities to act as aprobiotic of the different strains of the invention were compared tothose observed in some commercial probiotic strains (Lactobacillusrhamnosus LGG from Valio, Lactobacillus johnsonii La1 from Nestk andLactobacillus casei immunitas from Danone) and quantificatedarbitrarially. TEST CECT5711 CECT5713 CECT5714 CECT5715 CECT5716 LGG LA1LC Adhesion to high moderate high high high high high slight Caco-2^(a)Adhesion to HT-29^(a) high high moderate moderate high high moderateslight Resistance to acid^(b) high moderate high moderate high moderateslight slight Resistance to bile^(b) high high slight slight moderateslight high high Time of generation^(c) moderate rapid moderate stowmoderate rapid moderate moderate API CH50^(d) slight high moderate highmoderate moderate moderate high Fermentation^(d) moderate high slightslight slight slight high high Antibiogram^(f) high slight moderate highhigh moderate high high Reduction of pH^(g) high moderate moderatemoderate moderate slight moderate slight Antimicrabiat prod.^(h) highhigh high (*) moderate moderate moderate(*) slight slight PathogenInhibition^(i) high high moderate high high high moderate slightProbiotic capability 26 28 23 23 26 23 24 20

Example 5 Probiotic Colonization of Mice Gut

[0154] By definition, a probiotic must colonize the gut mucosa of thehost. Moreover, it has been described that the beneficial actionsexerted by probiotics require this colonization. Although in vitrostudies such as adhesion capabilities to intestinal cell lines, orresistance to the digestion conditions are good approaches to selectprobiotic strains, these tests do not ensure the effectiveness of theselected strain to colonize in vivo the gut mucosa. For this reason, weperformed an analysis in vivo of the colonization capacity of theprobiotic strains of the invention using mouse as an experimental animalmodel.

[0155] Six male Balb/c mice (6-8 weeks old) were daily supplemented with10⁸ cfu in 0.2 ml of skimmed milk of L. salivarius CECT5713 for 14 days.After this period, the probiotic supplementation was stopped but theanimals were still kept in observation for another 14 days. Fecessamples were collected at 0, 7, 14, 21 and 28 days from the initiationof the experience. Aprox. 200 mg of feces were collected independentlyfrom each mice and homogenized at 50 mg/ml in peptone water. Serialdilutions of the collected supernatant were prepared, and 0,1 ml platedin selective agar plates (MRS for Lactobacilli, Eugon agar+tomato juicefor Bifidobacteria and McConkey agar for coliform bacteria). Plates wereincubated at 37° C. in anaerobic conditions for 24 hours. The number ofcfu was determined by counting on selective media plates and the veragewas calculated.

[0156]FIG. 13 shows that supplementation with L. salivarius CECT5713caused a stadistically significant increase in the number of totallactobacilli in feces which demonstrates that this strain is able tosurvive its passage through the digestive tract and reach the colon.Moreover, the fact that the increased lactobacilli count was stillobservable one week after finalization of the oral supplementationdemonstrates that this probiotic strain is able to temporally colonizethe gut mucosa.

[0157] Concomitantly with the lactobacilli increase, a reduction in thefecal count of coliform bacteria was also observed, and was stillstatistically significant two weeks after finalization of the probiotictreatment. These findings show that dietary supplementation withCECT5713 cells causes not only stimulation of the beneficial flora butalso inhibition of the harmful bacteria.

Example 6 Effect of Lactobacillus fermentum CECT5716 on translocation ofSalmonella typhimurium in Mice Following Immunization with InactivatedSalmonella Vaccine

[0158] Translocation of Gram-negative bacteria across the gut epitheliumcan occur especially in subjects following gastrointestinal infection,disease or surgery. Left untreated it can lead to endotoxemia. In thisexample, the effect of feeding L. fermentum CECT5716 on thetranslocation of gut pathogen Salmonella typhimurium was examined.

[0159] Male Balb/c mice (6-8 weeks old) were daily orally inoculatedwith 1×10⁸ cfu in 0.2 ml of milk or milk alone for two weeks. Afterthat, mice were immunized orally or not with an inactivated Salmonellavaccine (10⁸ cfu inactivated with paraphormaldehyde in 0.2 ml milk).After immunization, mice were orally inoculated two weeks more with theL. fermentum CECT5716 preparation in alternate days for two weeks more.Two weeks after oral immunization, all mice were orally challenged withlive S. typhimurium (10¹⁰ cfu in 0.2 ml milk). Then, after 24-48 hours,the level of colonization of S. typhimurium in the spleen was determinedby colony counting in SS agar (Oxoid). The fecal concentration of IgAspecific for Salmonella antigens were also measured by ELISA techniques(Biosource).

[0160] The results obtained demonstrate that L. fermentum CECT5716potentiates the beneficial effect of the vaccination of mice with theinactivated Salmonella vaccine as shown in FIG. 15. The inhibition onthe translocation of S. typhimurium induced by the inactivated vaccineand potentiated by L. fermentum CECT5716 was due to the increase of thesecretion of specific IgA and also, to the inhibition or blocking effectof the probiotic strain on the mucosal adhesion of Salmonella asdescribed in Example 4h.

Example 7 Effect of Probiotic Bacteria on Inflammatory Cytokines

[0161] Besides the reduction of the risk of infection, many clinicaleffects associated to probiotic treatments are due to immuno-modulatorycapabilities of selected probiotic strains. The regulation of the immuneresponse is usually mediated through a change in the balance betweenpro-inflammatory cytokines (Th1) such as TNF-α, humoral cytokines (Th2)such as IL-4 or IL-13, and regulatory cytokines (Th3) such as IL-10 andTGF-β. For this reason, the effect of some of the probiotic strains ofthis invention in regulating the expression of some of these crucialcytokines was also tested.

[0162] Bone marrow derived macrophages were stimulated with 100 ng/ml ofLPS (Sigma) as a cellular model. 10⁵ macrophages/well were cultured in24-well plastic plates (Nunc) with 1 ml of DMEM. Once attached,macrophages were stimulated or not with 100 ng/ml LPS and with 10⁷cfU/ml of the indicated probiotic strains for 12 hours at 37° C. in a 5%CO₂ atmosphere. Supernatants were collected and the production ofcytokines was analyzed using a mouse TNF-α or mouse IL-10 ELISA(Biosource).The results obtained (FIG. 15) show that the consumption ofthe probiotic strains of this invention could have a beneficial effectin some inflammatory situations since they induce a globalanti-inflammatory effect on immune cells such as macrophages, inducingan increase in IL-10 expression without increasing the levels ofsecreted TNF-α.

Example 8 Effect of Probiotic Bacteria on Ig Production

[0163] The effect of the probiotic strains of this invention on theimmunoglobulin production was analyzed using lymphocyte culturesobtained from the spleen of male Balb/c mice (6-8 weeks old). 2×10⁶lymphocytes were cultured in 1 ml DMEM in 24 well plastic plates andstimulated with inactivated probiotic cultures (10⁸ cfu/ml) in presenceor absence of 25 μg/ml LPS for 6 days. The production of Ig G bylymphocytes was assessed using a mouse Ig G ELISA from Bethyl.

[0164] The results obtained (FIG. 16) show that the effect on the Ig Gproduction induced in lymphocytes of the probiotic strains of thisinvention is highly variable depending on the selected strain used. Inthis regard, there is some strains (CECT5711 and CECT5714) that haveimmune-stimulating activities since induce the expression of Ig G, whileothers (CECT5713 and CECT5715) have immune-suppressive effects.

Example 9 Preparation of a Fermented Liquid Milk Formula

[0165] A normal fermented liquid milk composition with probiotics wasprepared using the following formula: Milk 1.5% Fat; 3.2% protein  997g/kg Skim milk powder   3 g/kg Probiotic strain (10¹² cft/g)  0.1 g/kg

[0166] The fat and dry solids contents of the milk were standardizedaccording to the formulation described above. After that, the milk washomogenized at 20-25 Mpa and 65-70° C. to obtain optimum physicalproperties in the product. The preparation was heated at 90-95° C. and aholding time of about 5 minutes. This period of time causes thedenaturation of about 70-80% of whey proteins. Cooled milk (40-45° C.)was inoculated with the probiotic strain in absence of any starterculture and fermented in the incubation tank at 40-45° C. for 10 hourswithout agitation until reaching a final pH (pH 4.5-5). After clotformation, is the mixture was homogenized by mechanical methods. Oncethe homogenization was carried out, the preparation was cooled down to atemperature below 10° C. in 60 minutes. After that, the composition waspackaged. Final cooling, normally down to 5° C., took place in a coldroom, where the products were held to caducity.

Example 10 Preparation of a Set Yogurts

[0167] A yogurt product with probiotics was prepared using the followingformula: Milk 3.1% Fat; 3.2% protein  987 g/kg Skim milk powder   13g/kg Starter  0.1 g/kg Probiotic strain (10¹² cfu/g)  0.1 g/kg

[0168] The fat and dry solids contents of the milk were standardizedaccording to the formulation described above. The milk was homogenizedat 20-25 Mpa and 65-70° C. to obtain optimum physical properties in theproduct and heat treatment was performed at 90-95° C. and a holding timeof about 5 minutes which is able to denature about 70-80% of wheyproteins. After pasteurization, milk was cooled to 40-45° C. and thestarter and probiotic cultures were metered into the stream of milk asthey were pumped from an intermediate storage tank to the fillingmachine. Following packaging in the filling machine, the packages aftercrating and palletizing, were trucked into the system for incubation andcooling. After that, filled pallets were fermented in the incubationroom at 40-45° C. for 5-6 hours until a pH of 4.5 was reached. Coolingof the packets were performed quickly obtaining a temperature of 12-15°C. in 55-70 minutes. Final cooling, down to 5° C., took place in thechill store.

Example 11 Preparation of an Infant Formula in Powder

[0169] An infant formula with probiotics was prepared using thefollowing formula: Demineralised whey 512 g/kg Palm olein 135 g/kgLactose  92 g/kg Skimmed Milk  95 g/kg Rapeseed oil  52 g/kg Coconut oil 49 g/kg Sunflower oil  28 g/kg Water  31 g/kg Vitamin premix  2 g/kgMineral premix  4 g/kg Probiotic strain (10¹² cfu/g)  0.1 g/kg 

[0170] To an appropriately sized blend tank with agitation and heatingall solid ingredients were mixed with the liquid milk and water in theabsence of any vitamins. Then, the vegetable oils were admixed. Themixture was then heated at 60-70° C. and emulsified through a singlestage homogenizer at 6 to 7 MPa in absence of oxygen. Afteremulsification the mixture was standardized by addition of vitamins andthe pH was adjusted in the range of about 6.7 to 7.2. Then, the mixturewas reheated to between about 65° C. and 70° C. The product was finallywas dried in a spray drier to obtain a final dry powder product.Finally, the probiotic strain (10¹² cfu/g)

[0171] (0.1 g/Kg) was dry mixed with the final dry powder product andwas packaged.

1. A method for the selection of probiotic microbial strains, comprisingthe following steps: a. selecting for non-pathogenic strains which arecapable of surviving in breast milk and/or amniotic fluid, and b.selecting for non-pathogenic strains which are able to be transferred tobreast milk and/or amniotic fluid after oral intake in healthyindividuals without colonizing other internal organs except mucousas. 2.A method according to claim 1, wherein both breast milk and amnioticfluid are from human sources.
 3. A method as claimed in claim 2, whereinthe probiotic tested strains are any lactic acid bacteria selected fromthe genera: Lactobacillus, Lactococcus, Leuconostoc, Enterococcus,Streptococcus and Bifidobacterium.
 4. A method according to claims 3,wherein the probiotic strains tested had previously been obtained from:breast milk, feces of breastfed babies or amniotic fluid.
 5. A methodaccording to claim 4, wherein the strains tested had been obtained fromhuman samples.
 6. A bacterial strain selected by a method according toany of claims 1 to 5
 7. A strain of bacteria deposited in the CECT underAccession N^(o) 5711 (Lactobacillus coryniformis) or a variant thereof.8. A strain of bacteria deposited in the CECT under Accession N^(o) 5713(Lactobacillus salivarius subsp. salivarius) or a variant thereof.
 9. Astrain of bacteria deposited in the CECT under Accession N^(o) 5714(Lactobacillus gasseri, formerly L. acidophilus) or a variant thereof.10. A strain of bacteria deposited in the CECT under Accession N^(o)5715 (Lactobacillus gasseri) or a variant thereof.
 11. A strain ofbacteria deposited in the CECT under Accession Nu^(o) 5716(Lactobacillus fermentum) or a variant thereof.
 12. A biologically pureculture of a strain according to any of claims 6-11.
 13. Use of mammalmilk and of mammal amniotic fluid as a source to obtain non-pathogenicprobiotic bacteria, especially probiotic bacteria selected from thegenera Lactobacillus, Lactococcus, Enterococcus, Streptococcus andBifidobacterium
 14. Use of mammal milk and of mammal amniotic fluidaccording to claim 13 where the mammal milk and the mammal amnioticfluid are human.
 15. Use of mammal milk and of mammal amniotic fluidaccording to claim 14 as a source to obtain the microbial strainsdefined in any of the claims 6 to
 11. 16. A composition comprising atleast one of the bacterial strains defined in claims 6 to 11, where thecomposition comprises preferably from 2 to 6 strains, more preferablyfrom 2 to 4 strains, most preferably from 2 to 3 strains, and where eachof the strains is present in the composition in a proportion from 0.1%to 99.9%, preferably from 1% to 99%, more preferably from 10% to 90%.17. A composition comprising at least one of the bacterial strainsdefined in claims 6 to 11 together with another strain or mixture ofstrains where the mixture comprises preferably from 2 to 6 strains, morepreferably from 2 to 4 strains, most preferably from 2 to 3 strains andwhere each of the strains is present in the composition in a proportionfrom 0.1% to 99.9%, preferably from 1% to 99%, more preferably from 10%to 90%.
 18. A composition comprising a strain as claimed in any one ofclaims 6 to 11 or a composition as defined in any of the claims 16 to 17in a lyophilized form.
 19. A composition comprising a strain as claimedin any one of claims 6 to 11 or a composition as defined in any of theclaims 16 to 17 in a frost form.
 20. A composition comprising a strainas claimed in any one of claims 6 to 11 or a composition as defined inany of the claims 16 to 17 in an inactivated form or dead.
 21. Acomposition obtainable from the supernatant of a culture of a bacterialstrain according to any of the claims 6 to 11, or from a compositionaccording to any of the claims 16 to
 17. 22. A composition obtainable byextraction of a culture of any of the bacterial strains according toclaims 6 to 11, or by extraction of a composition according to claims 16to
 17. 23. A product obtainable from the metabolic activity of any ofthe strains specified in claims 6 to 11, from a culture according toclaim 12 or from a composition according to claims 16 to 17, wherein theproduct is preferably an enzyme.
 24. Food product comprising supportmaterial and at least one strain according to any of the claims 6 to 11,a culture, a composition or a product according to claims 12 and 16 to24.
 25. A product according to claim 24 wherein the support material isa food composition selected from milk, yoghourt, curd, cheese, fermentedmilks, milk based fermented products, meat based fermented products,fermented cereals based products, milk based powders, cereal basedpowders, infant formulae, clinical nutrition formula, ice-creams,juices, flours, bread, cakes, sugar, candies or chewing-gums.
 26. Aproduct according to claim 24, wherein the microbial strain according toclaims 6 to 11 is contained in the support material in an amount fromabout 10⁵ cfu/g to about 10¹² cfu/g support material, preferably fromabout 10⁶ cfu/g to about 10¹¹ cfu/g support material, more preferablyfrom about 10⁶ cfu/g to about 10¹⁰ cfu/g support material.
 27. Apharmaceutical composition comprising at least one strain according toany of the claims 6 to 11, a culture, a composition or a productaccording to claims 12 and 16 to 26 and pharmaceutically acceptableexcipients.
 28. A pharmaceutical composition according to claim 27wherein the microbial strains are contained in an amount from 10⁵ cfu/gto 10¹⁴ cfu/g support material, preferably from 10⁶ cfu/g to 10¹³ cfu/gsupport material, more preferably from 10⁷ cfu/g to 10¹² cfu/g supportmaterial.
 29. A strain of bacteria as claimed in anyone of claims 6 to11, a culture, a composition or product according to claims 12 and 16 to26 for therapeutic or prophylactic treatment.
 30. A compositionaccording to claim 29 for topic, oral, ocular, nasal, enteral,urogenital, vaginal or rectal administration
 31. A composition accordingto claim 29 designed to be administered to pregnant woman for thetherapeutic or prophylactic treatment of their foetus.
 32. A compositionaccording to claim 29 designed to be administered to lactating woman forthe therapeutic or prophylactic treatment of their breastfed babies. 33.Use of a strain according to any of the claims 6 to 11, a culture, acomposition or a product according to claims 12 and 16 to 27 in themanufacture of a product for the therapeutic or prophylactic treatmentof human and animal diseases.
 34. Use according to claim 33 for thetreatment and/or prophylaxis of chronic or acute infection, or ofundesirable microbial colonization, wherein the infection orcolonization is caused by parasites, bacteria, yeasts, fungi or viruses,of a mucosal surface in a subject or animal in need thereof, wherein themucosal surface is selected from but not restricted to the groupconsisting of oral, nasopharyngeal, respiratory, gastric, intestinal,urogenital and glandular.
 35. Use according to claim 33 for thetreatment and/or prophylaxis of temporally depressed immune levels inindividuals subjected to physiological stress.
 36. Use according toclaim 33 for the improvement of the immune gut barrier in a subject oranimal in need thereof; for the treatment and/or prophylaxis ofdown-regulating hypersensitivity reactions to food and metabolicintolerance such as: lactose intolerance; of constipation and othergastro-intestinal disorders; of inflammatory or auto-immune disorderssuch as: 113D, ulcerative colitis, arthritis, atherosclerosis, multiplesclerosis, psoriasis or sarcoidosis; and of tumor growth, metastasis andcancer in a subject or animal in need thereof.
 37. Use according toclaim 33 for the treatment and/or prophylaxis of allergic disorders andasthma in a subject or animal in need thereof.
 38. Use according toclaim 33 for the treatment and/or prophylaxis of neuro-degenerativediseases in an individual or animal in need thereof, selected from, butnot restricted to, the group consisting of Parkinson, stroke, Alzheimer,Huntington and dementia.